This project proposes to investigate in vitro the sources of urinary ammonia nitrogen. Of particular interest is the possibility that amino acids other than glutamine are important nitrogen donors to renal ammonia, depending upon acid-base status. The influence of pH on ammonia formation from either the amide or alpha nitrogens of glutamine is also a target of study. Emphasis is placed on the respective roles of the purine nucleotide cycle and the glutamate dehydrogenase reactions as metabolic pathways of renal ammoniagenesis. The relationship between ammoniagenesis and the metabolism of alpha-ketoglutarate, particularly the role this organic acid plays in stimulating ammonia formation from glutamate, is another investigative focus. Experiments will be performed in rat kidney cortex slices, isolated renal tubules, kidney mitochondria, and the isolated perfused kidney. A unique feature of the project is the utilization of 15N as a tracer for renal nitrogen metabolism and the utilization of gas chromatography-mass spectrometry to quantitate 15N levels in this tissue. The data so generated will be of scientific value in improving our understanding of renal nitrogen metabolism.